Mosaicism of Activating FGFR3 Mutations in Human Skin Causes Epidermal Nevi

Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi

Christian Hafner, … , Ellen C. Zwarthoff, Arndt Hartmann

J Clin Invest. 2006;116(8):2201-2207. https://doi.org/10.1172/JCI28163.

Research Article Dermatology

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

Find the latest version: https://jci.me/28163/pdf Research article Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi Christian Hafner,1 Johanna M.M. van Oers,2 Thomas Vogt,1 Michael Landthaler,1 Robert Stoehr,3 Hagen Blaszyk,4 Ferdinand Hofstaedter,5 Ellen C. Zwarthoff,2 and Arndt Hartmann5

1Department of Dermatology, University of Regensburg, Regensburg, Germany. 2Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands. 3Department of Urology, University of Regensburg, Regensburg, Germany. 4Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, USA. 5Institute of Pathology, University of Regensburg, Regensburg, Germany.

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

Introduction domains (Figure 2). Alternative splicing of the second half of the Epidermal nevi show a prevalence of about 1 in 1,000 people and IgIII-like domain (exon 8 versus exon 9) results in the isoforms can be divided into either nonorganoid (keratinocytic) types or IIIb and IIIc. The isoforms show different ligand specificity and organoid types characterized by hyperplasia of adnexal structures tissue expression. FGFR3 IIIb is mainly expressed in epithelial cells such as sebaceous glands, sweat glands, and hair follicles. Epider- while FGFR3 IIIc is predominantly found in mesenchymal cells (5). mal nevi of the common, nonorganoid and nonepidermolytic type This paper assigns all codon numbers to the open reading frame of are benign skin lesions and may vary in their extent from a single the FGFR3 IIIb isoform. More than 20 FGFs are known as ligands (usually linear) lesion to widespread and systematized involvement now (4). The interaction of the ligand with the receptor requires (Figure 1). They may be present at birth or develop early during the presence of sulfated glycosaminoglycans such as heparin and childhood as localized epidermal thickening with hyperpigmenta- leads to the dimerization of the receptor with consecutive phos- tion, frequently following the lines of Blaschko. This suggests that phorylation of intracellular tyrosine residues in the kinase domain epidermal nevi may be due to mosaicism resulting from postzygot- and activation of intracellular signaling pathways. The autophos- ic mutations in keratinocytes. Mutations of keratins 1 and 10 were phorylation sites of FGFR3 represent potential binding sites for shown to be responsible for a rare subgroup of epidermal nevi, signaling proteins, for example, with phosphotyrosine binding the linear epidermolytic hyperkeratosis (1, 2). Another variant of (PTB) and Src homology 2 (SH2) domains. Activation of the cyto- epidermal nevi, the congenital hemidysplasia with ichthyosiform plasmic region causes phosphorylation of Shp2, PLCg, ERK1/2, nevus and limb defects (CHILD) nevus, is caused by NADPH ste- and STAT3 and also PI3K activation (4). FGFRs can also be acti- roid dehydrogenase-like protein (NSDHL) mutations (Xq28) and vated by interaction with EphA4, another receptor tyrosine kinase, represents a functional X chromosomal mosaicism (3). However, which demonstrates the complexity of FGFR signaling (6). the genetic basis of the much more common nonorganoid, non- Activating germline mutations of the FGFR3 gene result in dwarf- epidermolytic keratinocytic epidermal nevi remains elusive. ism, severe skeletal dysplasia, and craniosynostosis syndromes such The FGF receptor (FGFR) family comprises 4 major trans- as achondroplasia (ACH), hypochondroplasia (HCH), thanato- membrane receptor tyrosine kinases (FGFR1–4) and is involved phoric dysplasia (TD), Crouzon syndrome (CS), Muenke syndrome in embryogenesis, angiogenesis, and tissue homeostasis (4). The (MS), and SADDAN (severe achondroplasia with developmental FGFR3 gene contains 19 exons encoding an extracellular region delay and acanthosis nigricans) syndrome (7–10). These activating for ligand binding composed of 3 Ig-like domains, a hydropho- mutations result in negative chondrocytic growth regulation of bic transmembrane domain and 2 cytoplasmic tyrosine kinase the epiphyseal plates of long bones, causing dwarfism (11). Identi- cal mutations are found in different cancer entities and probably provide proliferative signals (4). FGFR3 signaling in mutated cells Nonstandard abbreviations used: ACH, achondroplasia; CS, Crouzon syndrome; is poorly understood, but previous studies provide some insights. FGFR3, fibroblast growth factor receptor 3; HCH, hypochondroplasia; SADDAN, severe achondroplasia with developmental delay and acanthosis nigricans; All known missense mutations causing TD I create an unpaired TD, thanatophoric dysplasia. cysteine residue (12–14), such as the R248C and S249C mutations. Conflict of interest: The authors have declared that no conflict of interest exists. These mutations are localized in the extracellular domain at the Citation for this article: J. Clin. Invest. 116:2201–2207 (2006). doi:10.1172/JCI28163. linker region between the Ig-like domains II and III while other

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Figure 1 Patient 29 displayed a systematized epidermal nevus of the common soft type with involvement of the face (bilateral), the right scapular region, the right arm, the right hip, and the right thigh. Abnormalities of the skeletal or nervous system were not present. A biopsy was taken from the epidermal nevus of the right forearm. This epidermal nevus revealed an R248C FGFR3 gene mutation. DNA isolated from the blood of this patient revealed WT status at codon 248, excluding a germline mutation.

to the formation of hydrogen bonds between 2 FGFRs, resulting in constitutive receptor activation (20). The A393E mutation in CS also affects the transmembrane domain and results in FGFR3 dimer stabilization, measured by the change in the free energy of the dimerization, thus largely increasing the fraction of dimers (21). A third class of mutations affect the tyrosine kinase domain, such as the K652E mutation in TD II or the N542K mutation in HCH (11). These mutations likely cause conformational changes in the activation loop that activate the receptor tyrosine kinase activity and downstream ERK1/2 (22). Other studies suggest that FGFR3 mutations may delay the downregulation and ligand-mediated internalization of the receptor (23). The phosphorylated immature form of the mutant receptor accumulates in the endoplasmic reticulum and fails to be degraded (24). The different degree of receptor activation seems to correlate with the severity of the phenotype. Stronger activation of the receptor by ligand-independent dimerization via disulfide bonds in TD patients deter- mines the more severe phenotype compared with other skeletal dysplasia syndromes such as ACH and HCH (15, 25). Somatic activating FGFR3 mutations have been identified in 40% of human seborrheic keratoses (26) and in several human cancers (4), including multiple myeloma (27), urothelial carcinoma (28), mutations causing unpaired cysteine residues such as G372C are cervix carcinoma (29), and colorectal carcinoma (30). Some of the localized at the junction of the extracellular and the transmem- skeletal dysplasia syndromes (TD, CS, SADDAN) caused by FGFR3 brane domain of FGFR3. The newly created unpaired cysteine mutations are also characterized by marked thickening of the epi- amino acid allows the formation of disulfide bonds between the dermis. This skin lesion, termed acanthosis nigricans, and epidermal extracellular domains of 2 receptors, resulting in homodimeriza- nevi share similar histological features, including acanthosis and tion, increased tyrosine phosphorylation, and ligand-independent papillomatosis (31, 32). Herein we investigate the role of FGFR3 constitutive receptor activation (15, 16). Similar mechanisms were mutations in common nonorganoid, nonepidermolytic keratino- also shown for other receptor tyrosine kinases, the erythropoietin cytic epidermal nevi. receptor and the epidermal growth factor receptor (17, 18). Con- stitutive activation in cells bearing the G372C or S373C mutation Results results in high basal phosphorylation with significantly increased We analyzed 39 common nonepidermolytic, nonorganoid kera- constitutive levels of MAPK phosphorylation and c-fos transcrip- tinocytic epidermal nevi of 33 patients using a SNaPshot mul- tion, probably caused by mutant homodimer FGFR3 complexes tiplex assay that covered 11 FGFR3 point mutations described (19). Other mutations, such as G382R, which is found in about in skeletal dysplasia syndromes and cancer entities (Figure 2). 97% of ACH patients, affect the transmembrane domain and lead The following subtypes of keratinocytic epidermal nevi were

Figure 2 FGFR3 gene. The position of the mutations covered by the SNaPshot multiplex assay is indicated. Codons are numbered according to the FGFR3 IIIb isoform; potential mutations of the stop codon 809 in exon 19 associated with TD I were analyzed by direct sequencing. C, C-terminus; Ig I, Ig II, Ig III, Ig-like domains I–III; N, N-terminus; TM, transmembrane domain; TKI, TKII, tyrosine kinase domains I–II.

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Table 1 FGFR3 mutations were found in common- FGFR3 mutations in epidermal nevi type epidermal nevi (14 common soft type, and 2 common hard type). No. Sex Age (yr) Site Subtype Nevus Control Syst. In 4 patients (patients 3, 17, 21, and 32) with 1 F 1 Head Seborrheic WT – No an FGFR3 mutation (R248C) in the epidermal 2 M 59 Head Acro-verruc WT – – nevus, clinically and histologically normal epi- 3 F 9 Left axilla Common hard R248C WT – dermis adjacent to the nevus showed a WT codon 4 M 13 Neck Seborrheic WT – No 248, suggesting an epidermal mosaicism of the 5 F 36 Right axilla Common soft R248C – No FGFR3 mutation and a strong genotype-pheno- 6 M 17 Head Common soft WT – – type correlation (Figure 3). In 1 patient (patient 7 M 7 Head Common soft WT – – 29) with a systematized epidermal nevus display- 8 M 21 Head Common soft WT – – ing the R248C mutation (biopsy was taken from 9 M 15 Head Common soft WT – – 10 F 15 Right shoulder Common hard WT – – the right forearm), additional genomic DNA was 11a M 22 Head Common soft WT – No isolated from blood. The DNA revealed the WT 11b M 22 Head Common soft WT – No codon 248, thus excluding a germline mutation. 12 M 20 Left groin Common soft R248C – No Multiple intraindividual epidermal nevi biop- 13 F 13 Back Common soft WT – – sies could be analyzed in 2 patients. One patient 14 F 23 Back Common soft WT – – (patient 11a, b) did not show any FGFR3 muta- 15 F 5 Left axilla Common soft G372C/G382R – – tions. The other patient (patient 33a–e) under- 16 F 2 Neck Common soft WT – No went ablative laser treatment of the common 17 M 6 Neck Common soft R248C WT Yes soft-type epidermal nevus at the right side of the 18 M 16 Head Common soft WT – No neck (Figure 4). Before treatment, 6 of the scat- 19 F 27 Neck Common soft WT – – 20 F 25 Right shoulder Common soft WT – – tered brownish papules were curetted for FGFR3 21 M 29 Head Common hard R248C WT No mutation analysis. All 6 samples, which were 22 F 20 Right arm Common soft R248C – – spatially distant from each other, revealed the 23 F 16 Trunk Common hard WT – No R248C mutation in the SNaPshot analysis. 24 M 31 Head Common soft WT – No All FGFR3 mutations detected so far in skin 25 F 18 Back Seborrheic WT – – lesions (seborrheic keratoses and epidermal nevi) 26 M 9 Neck Common soft R248C – Yes are associated with TD, CS, and SADDAN syn- 27 M 14 Head Seborrheic WT – No drome in the germline. The SNaPshot multiplex 28 F 9 Head Acro-verruc WT – No assay covered all mutations responsible for these 29 F 11 Right forearm Common soft R248C – Yes syndromes except for the stop codon mutation 30 F 15 Head Common hard WT – No 31 F 16 Neck Common soft WT – – X809L/G/R/C/W in exon 19 causing TD I (Fig- 32 M 23 Back Common soft R248C WT No ure 2). For 20 epidermal nevi (nevi of patients 1, 33a F 13 Neck Common soft R248C – Yes 2, 5, 6, 7, 8, 9, 10, 11 [a, b], 14, 16, 17, 20, 23, 27, 33b F 13 Neck Common soft R248C – Yes 28, 30, 31, and 32), DNA was available for fur- 33c F 13 Neck Common soft R248C – Yes ther analysis. We additionally sequenced exon 19 33d F 13 Neck Common soft R248C – Yes of these samples. Seventeen of the 20 epidermal 33e F 13 Neck Common soft R248C – Yes nevi had not shown any FGFR3 mutations in the 33f F 13 Neck Common soft R248C – Yes SNaPshot analysis. However, no further FGFR3 Age, age at time of biopsy; site, site of biopsy; control, DNA from clinically and histologically mutations were detected in exon 19. normal epidermis adjacent to the epidermal nevus; syst., systematized epidermal nevus; Since epidermal nevi represent a heteroge- M, male; F, female; acro-verruc, acrokeratosis verruciformis-like; R248C, exon 7, codon 248, neous skin disorder, we also studied an organ- CGC to TGC with Arg to Cys; G372C, exon 10, codon 372, GGC to TGC with Gly to Cys; oid type of epidermal nevus, the sebaceous G382R, exon 10, codon 382, GGG to AGG with Gly to Arg. Codon numbers refer to the open reading frame of the FGFR3 IIIb isoform. nevus. This nevus is found almost exclusively on the scalp or face and is usually present at birth. Sebaceous nevi are histopathologically included in the study: 28 common soft type, 5 common hard characterized by the presence of large numbers of mature seba- type, 4 seborrheic-like type, and 2 acrokeratosis verruciformis- ceous glands and papillomatous hyperplasia of the epidermis. We like type. We identified activating FGFR3 mutations in 11 of 33 analyzed 13 sebaceous nevi for FGFR3 point mutations using the (33%) patients and in 16 of 39 (41%) epidermal nevi (Table 1). In SNaPshot multiplex assay (Table 2). In contrast to the common 15 of 16 epidermal nevi (94%), the mutation occurred in exon 7 nonorganoid keratinocytic nevi, this organoid type of epidermal at codon 248 (CGC to TGC), resulting in an Arg to Cys amino nevus revealed no FGFR3 mutations. Thus, activating FGFR3 muta- acid substitution. In one epidermal nevus (patient 15), a double tions, especially the frequently detected R248C mutation, appear mutation was found in exon 10 at codon 372 (GGC to TGC), to be specific for common nonorganoid keratinocytic nevi. resulting in a Gly to Cys amino acid substitution, and at codon 382 (GGG to AGG), resulting in a Gly to Arg amino acid sub- Discussion stitution. The epidermal nevus with the double mutation was Our results indicate that a significant number of epidermal nevi a common soft-type nevus and showed no histological abnor- of the common, nonorganoid, and nonepidermolytic keratinocytic malities compared with the other epidermal nevi. All activating type are caused by postzygotic mutations in the FGFR3 gene, which

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show acanthosis, papillomatosis, hyperkeratosis, and basal hyperpigmentation (31, 32). A potential overlap between the 2 skin disorders is acknowledged since both acanthosis nigricans type of epidermal nevus and nevoid acanthosis nigricans have been described (32, 35, 36). In our series of epidermal nevi, an acanthosis nigricans-like type was not included. A female patient with a mosaicism of R248C (25% of the blood lymphocytes were affected by the mutation) developed disseminated thickening and hyperpigmentation of the skin consistent with acanthosis nigricans (37). (d) Some patients with epidermal nevus syndrome showed typical keratinocytic epidermal nevi, skeletal abnormalities, and the occurrence of urothelial carcinoma at an early age (38–40). This correlation of epidermal nevus and urothelial Figure 3 carcinoma is thought to be nonstochastic. FGFR3 muta- Patient 32 had a common soft-type epidermal nevus on his back. Two biop- tions are frequent events in papillary urothelial carcinoma sies were taken from the epidermal nevus and the adjacent normal skin. (29, 41). These findings and the skeletal abnormalities rem- The epidermal nevus histologically showed the typical acanthosis and papil- iniscent of skeletal dysplasia syndromes strongly suggest lomatosis (H&E staining; original magnification, ×40) and an R248C muta- that such patients feature a mosaicism of activating FGFR3 tion in the SNaPshot analysis. In contrast, the clinically and histologically mutations, which in turn cause epidermal nevi of the skin, normal epidermis revealed a WT status for codon 248. This result suggests a strong genotype-phenotype correlation and the presence of a mosaicism skeletal changes, and bladder cancer. (e) In one patient of of the FGFR3 mutation in the epidermis of this patient. our series (patient 33), multiple intraindividual samples of the epidermal nevus localized on the neck could be analyzed. All 6 samples revealed the same R248C mutation, suggest- likely occur early in embryonic development in a single keratinocyt- ing the presence of a scattered FGFR3 mutation mosaicism in the ic stem cell. The descendants of the mutated stem cell will migrate skin of this patient following the lines of Blaschko. (f) The stron- along the lines of Blaschko, resulting in the linear or whirled cuta- gest support that the detected mutations are indeed causative for neous patterns usually observed in epidermal nevi (33). epidermal nevi is that 4 patients with an R248C mutation in the Several findings support the notion that the detected FGFR3 epidermal nevus showed the WT allele in clinically and histologi- mutations are causative for the development of epidermal nevi: cally normal epidermis adjacent to the epidermal nevus. This also (a) The detected mutations R248C and, in one case, G372C are suggests mosaicism of an FGFR3 mutation in the epidermis and a known to occur in TD I patients and act as dominant germline strong genotype-phenotype correlation. mutations in this lethal skeletal dysplasia syndrome. Affected In our series, 15 of 16 epidermal nevi with an FGFR3 mutation individuals are often stillborn or die as neonates. It has previously displayed the R248C mutation, resulting in a cytosine-thymine been postulated that such lethal mutations may only survive by substitution (CGC to TGC). This C to T transition is a typical mosaicism (34). The R248C mutation results in strong constitu- example of deamination of methylated cytosines leading to CpG tive and ligand-independent receptor activation (15), excluding dinucleotide depletion. The reason for this mutational hot spot the possibility that the detected mutations have no functional is unknown. One could speculate that the development of epi- consequence in the investigated skin samples. (b) Mutations responsible for TD I (including R248C and G372C) have been shown to cause another benign skin lesion, the seborrheic kerato- sis (26). Epidermal nevi and seborrheic keratoses share many clinical and histopathological features. Another activating mutation (S249C) associated with TD I causes thickening of the skin and verrucous skin tumors in a mouse model. The murine skin lesions resemble seborrheic keratoses (and likewise epidermal nevi) clinically and histologically (26). (c) A TD I patient with an R248C mutation and long-term survival developed acanthosis nigricans, a skin disorder that is clinically and histologically very similar to epidermal nevi (10). Both acanthosis nigricans and epidermal nevi

Figure 4 Patient 33 revealed a common soft-type epidermal nevus at the right side of the neck consisting of scattered brownish papules following the lines of Blaschko. Before ablative laser treatment, 6 papules distant from each other were curetted for FGFR3 mutation analysis. The green peaks represent WT codon 375, the black peaks WT codon 248. The R248C mutation is characterized by a red peak. All 6 samples displayed the R248C mutation. In the bottom right corner, control DNA with WT codon 248 is shown.

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Table 2 syndromes. The reason for this preferential localization remains FGFR3 mutation analysis in sebaceous nevi unclear since the entire skin carries the mutation. There seems to be a preponderance of intertriginous localization (neck, axilla, No. Sex Age (yr) Site Sebaceous nevus groin) also in the epidermal nevi with FGFR3 mutations in our 1 M 17 Head WT series (Table 1). Additional cofactors in intertriginous areas may 2 M 15 Head WT favor the development of acanthosis and papillomatosis on the 3 F 7 Head WT basis of activating FGFR3 mutations. 4 M 12 Head WT One epidermal nevus in our series displayed a double mutation, 5 M 33 Head WT the G372C mutation known from TD I and the typical G382R 6 M 17 Head WT mutation known from ACH. It remains elusive whether the G372C 7 M 7 Head WT mutation alone, which causes a stronger activation of the FGFR3 8 M 14 Head WT receptor according to the associated skeletal dysplasia syndromes 9 M 13 Head WT 10 F 16 Head WT (13–15), was able to cause the epidermal nevus or whether the 11 M 12 Head WT combination of both mutations was necessary for the induction 12 M 32 Head WT of the nevus. The possibility that the nevus was mainly caused by 13 M 13 Head WT the G382R mutation seems unlikely since ACH patients, in contrast to TD patients, usually do not develop acanthosis nigricans (10, 42). Double mutations in the FGFR3 gene have been reported in urothelial carcinoma (28, 47, 48). To our knowledge, germline dermal nevi with acanthosis and papillomatosis of the epidermis compound heterozygosity for mutations associated with TD and requires strong activation of the receptor and not all activating ACH has not been described. The epidermal nevus carrying the FGFR3 mutations are capable of this strong activation. Indeed, double mutation displayed no histopathological abnormalities. Naski et al. found that the R248C mutation activates the receptor Each of the major 3 cell types present in our microdissected more strongly than the G382R mutation responsible for ACH (15). samples (keratinocytes, dendritic cells, and melanocytes) may Stronger activation of the receptor is thought to result in a more theoretically be the carrier of the detected mutations, but kera- severe phenotype in TD compared with other skeletal dysplasia tinocytes represent the likely host cell type. Logie et al. studied a syndromes (14). According to this theory, mosaicism of FGFR3 mouse model in which the S249C mutation (causing TD I) was mutations other than R248C may also be present in the skin, but targeted to the basal layer of the epidermis using the keratin 5 pro- they may be insufficient to induce epidermal nevi. This hypoth- moter (26). Keratin 5 is a marker for basal keratinocytes and not esis is supported by Logie et al. (26), who found that seborrheic expressed in melanocytes or dendritic cells. The transgenic mice keratoses sharing some histological characteristics with epidermal developed thickening of the skin and verrucous skin tumors with nevi are caused by acquired somatic FGFR3 mutations. All somatic histological features similar to epidermal nevi. The basal hyper- mutations detected so far in seborrheic keratoses (R248C, S249C, pigmentation mediated by melanocytes would be a secondary phe- G372C, S373C, Y375C, K652E, and K652M) are associated with nomenon if the mutations affect keratinocytes. However, further TD and SADDAN syndrome in germline. Remarkably, none of studies are needed to prove this hypothesis. the mutations associated with ACH or HCH were found in this It is unclear why the same mutation (R248C) can cause both series of 62 seborrheic keratoses. Patients with TD and SADDAN seborrheic keratoses and epidermal nevi. FGFR3 mutations exhibit syndrome show acanthosis nigricans whereas ACH and HCH are pleiotropic effects ranging from inherited skeletal dysplasia syn- usually not associated with this skin lesion except for in 1 reported dromes and benign skin tumors to cancer. The cell type–specific case (42). ACH is the most common cause of dwarfism in humans, involvement of different signaling pathways such as Ras/MAPK with an incidence of 1:15,000 to 1:40,000 of live births, and the and STAT as well as the FGFR3-dependent recruitment of cell-spe- mutated nucleotide 1138 in ACH is thought be the most muta- cific second receptors such as EphA4 (6) may determine the effect ble nucleotide in the human genome described so far (9, 43). The of activating FGFR3 mutations in each cell type (4). R248C mutation is the most common mutation in TD (44), but Mutations of FGFR3 in the urothelium are significantly associ- it remains elusive why other mutations causing TD and SADDAN ated with benign urothelial papillomas (49) and with low-grade syndrome (S249C, S373C, Y375C, K652E, K652M) were not and low-stage pTa G1/2 tumors, which rarely progress (41, 48, detected in our series of epidermal nevi. 50). These findings support the concept that mutations of the Epidermal nevus syndromes are characterized by epidermal FGFR3 IIIb isoform can induce proliferation and tumor forma- nevi, abnormalities of the skeletal and nervous system, and tion but are associated with a low malignant potential. This rarely, some associated cancer entities (45, 46). The occurrence would be consistent with our findings of FGFR3 mutations in of papillary bladder cancer and skeletal abnormalities in epider- epidermal nevi that represent a benign skin disorder and show mal nevus syndromes (38–40) suggests that FGFR3 is a promis- signs of hyperproliferation (namely acanthosis and papilloma- ing candidate gene for epidermal nevus syndromes that may be tosis) but bear no malignant potential. caused by a more widespread mosaicism of FGFR3 mutations. If Several small molecule tyrosine kinase inhibitors of FGFR3, such the FGFR3 mosaicism involves the germ cells in those patients, as PKC412, PD173074, and SU5402, are already available. They the offspring should show a TD phenotype. However, we did not have been used in vitro and in animal models to inhibit the growth find any reports of patients with systematized epidermal nevi or of multiple myeloma cell lines with activating FGFR3 mutations epidermal nevus syndrome and an offspring with TD in the litera- (51–54). PKC412 is currently being evaluated in phase II trials for ture. Another interesting fact is that acanthosis nigricans is pre- acute myeloid leukemia patients (55). This molecule effectively dominantly observed in intertriginous areas in skeletal dysplasia inhibits the tyrosine kinase activity of FGFR3, as shown by the

The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 2205 research article inhibition of proliferation in hematopoietic cells transformed by ing to the manufacturer’s protocol. The DNA of each tissue was eluted in a a mutant FGFR3 gene (55). A broader use of this drug in other volume of 200 ml elution buffer. The amount of isolated DNA ranged from disorders associated with an increased activity of FGFR3 has been 5 mg to 30 mg for each sample. suggested. This may also include benign skin lesions such as seb- SNaPshot assay. A previously described SNaPshot multiplex assay, based orrheic keratoses (26) and epidermal nevi. The current standard on the SNaPshot Multiplex System assay (Applied Biosystems), was used to therapy for epidermal nevi and seborrheic keratoses is surgery or screen for activating FGFR3 point mutations (56). We used 2 ml template ablative laser treatment, which is often associated with irreversible DNA for the multiplex PCR. The SNaPshot multiplex PCR assay can detect scar formation. Topical application of tyrosine kinase inhibitors of mutations with an input DNA amount of only 1 ng genomic DNA (56). FGFR3 may obviate the need for surgical intervention. Three regions of interest in exons 7, 10, and 15 comprising 11 FGFR3 muta- In summary, a large proportion of human epidermal nevi are tions were amplified in 1 multiplex PCR, followed by extension of muta- caused by mosaicism of postzygotic activating FGFR3 mutations tion-specific primers with a labeled dideoxynucleotide. Two new antisense in the human epidermis. The R248C mutation appears to be a hot primers were added to the original assay to screen for mutations S373C spot for FGFR3 mutations in epidermal nevi. Additional studies (5′-T19GAGGATGCCTGCATACACAC-3′) and G382R (5′-T56GAACAG- are needed to investigate other regions of FGFR3 and different GAAGAAGCCCACCC-3′). Concentrations for those primers used in the receptor tyrosine kinases as possible mutational targets in epi- multiplex assay were 1.0 and 0.6 pmol/ml, respectively. Thus, screening dermal nevi and elucidate functional aspects of enhanced FGFR3 could be performed for 11 known mutations found in bladder tumors and signaling in the skin. other noncutaneous epithelial malignancies (R248C, S249C, G372C, S373C, Y375C, G382R, A393E, K652E, K652M, K652Q, and K652T; codons are Methods numbered according to the open reading frame of the FGFR3 IIIb isoform, Microdissection. Thirty-nine histologically confirmed nonepidermolytic which is predominantly present in epithelial cells). This mutation spectrum epidermal nevi of the common nonorganoid type of 33 patients with also covers the most frequently found FGFR3 mutations in skeletal dysplasia varying degrees of skin involvement were retrieved from the histol- syndromes. Extended primers were separated by capillary electrophoresis in ogy files of the Department of Dermatology, University of Regensburg. an automatic sequencer, and the presence or absence of a mutation was indi- Informed consent for the scientific use of the material and photographs cated by the incorporated WT or mutant labeled dideoxynucleotide. When a had been obtained from all patients according to the guidelines of the mutation is present, a second peak from the mutated nucleotide will appear ethics committee of the University of Regensburg and the Declaration of next to the WT peak in the electropherogram (see Figures 3 and 4). However, Helsinki. Classical symmetrical acanthosis nigricans in addition to the the assay is not quantitative due to the different emission efficiencies of the epidermal nevi was not seen in any of the study patients, and no signs labels. Mutations were confirmed by a second independent reaction. of skeletal dysplasia or associated cancer were observed. We classified FGFR3 sequence analysis. In addition to the SNaPshot analysis, exon 19 of the different subtypes of epidermal nevi according to a previous study the FGFR3 gene was directly sequenced. This exon contains the potential (32). The common keratinocytic type was subdivided into soft and hard mutation at stop codon 809 associated with TD I. We were able to analyze types according to the degree of hyperkeratosis. The characteristics of 20 epidermal nevi. The other samples failed due to limited DNA amounts. the patients and their nevi are shown in Table 1. We also investigated an We used the forward primer 5′-CCTGTCGGCGCCTTTGGAGCAG-3′ and organoid epidermal nevus type, the sebaceous nevus, because epidermal the reverse primer 5′-CAGACCAAAGCTCTGTAGCT-3′ to generate a 235 nevi represent a heterogeneous group of lesions. Thirteen sebaceous nevi bp PCR product of exon 19 containing the stop codon 809. Sequence anal- were retrieved from the histology files of the Department of Dermatology ysis was performed following standard protocols. of the University of Regensburg (Table 2). Sections of 10-mm thickness were microdissected manually from par- Acknowledgments affin-embedded epidermal nevi tissues with a needle under an inverted We thank our patients for their participation and Alois Eckl for microscope. We dissected the acanthotic epidermis of the nevi containing the helpful support. The excellent technical work of Monika Ker- mainly keratinocytes but also small numbers of melanocytes and dendritic scher, Kirstin van der Keur, Anne Pietryga-Krieger, Lydia Kuenzel, cells. In sebaceous nevi, both the hypertrophic sebaceous glands and the and Nadine Wandtke is gratefully acknowledged. acanthotic epidermis were dissected. Clinically and histologically normal epidermis adjacent to the nevus was microdissected in 4 patients with com- Received for publication February 8, 2006, and accepted in revised mon keratinocytic nevi to serve as a source of control DNA. form May 16, 2006. DNA isolation. DNA was isolated following standard protocols. In brief, about 25–50 mg formalin-fixed paraffin-embedded tissue was microdis- Address correspondence to: Christian Hafner, Department of Der- sected for each sample. The microdissected tissue was digested with pro- matology, University of Regensburg, Franz-Josef-Strauss-Allee 11, teinase K overnight in lysis buffer, and DNA isolation was performed with 93042 Regensburg, Germany. Phone: 49-941-944-9610; Fax: 49- the High Pure PCR Template Preparation Kit (Roche Diagnostics) accord- 941-944-9611; E-mail: [email protected].

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